Analytical Sciences, Talk
Capillary zone electrophoresis as a capable tool in endotoxin and carbohydrate analysis
Blanka Bucsella1, Antoine Forange1, Mònica Bassas1, Franka Kálmán1
1University of Applied Sciences and Arts Northwestern Switzerland, FHNW
Biotechnological products manufactured by Gram-negative bacteria and consumed by humans or animals must be proven to be endotoxin free in order to prevent endotoxic shock, inflammation and / or sepsis. During the development of the production process, determining the endotoxin levels is important to assess the efficiency of endotoxin removal methods. The widely used, FDA approved LAL (Limulus Amebocyte Lysate) assay is both time consuming and expensive, especially during the development of endotoxin removal methods due to its high error and small dynamic range, therefore a low cost method would be beneficial. From the chemical structure point of view endotoxins are complex lipopolysaccharides that show high biodiversity, and they are lacking the UV active moieties. Considering these facts two methods are adapted and optimized for their quantitative and qualitative analysis.
In order to determine the total endotoxin content in biopolymers produced by Gram-negative bacteria, based on [1], we have installed and optimized an indirect-UV detection method utilizing CZE-DAD. In the high UV absorbing background electrolyte (BGE), proteins and nucleotides of the crude samples are not detected meanwhile non-UV absorbing native endotoxins provide negative peaks. Due to their specific electrophoretic mobilities endotoxins are sufficiently separated from other non-UV absorbing species like carbohydrates or small ions. Endotoxin standards from different biological sources and of different purity are analyzed; results are compared to each other and to the LAL assay.
For the characterization of the diversity of the polysaccharide (O-antigen) part of the different endotoxins, an on-column reaction based CZE-DAD method was installed and optimized [2]. Liposaccharides or after quantitative hydrolysis the respective monosaccharaides are separated at high pH (12.6). Employing a DAD in the capillary detection window, the carbohydrates undergo an on-column reaction resulting in UV active species which are detectable. They are detected by direct UV-detection at 270 nm with a LOQ of about 10 µg / ml. Detection sensitivity is improved with the application of cationic surface modifiers in the background electrolyte.
[1] Freitag R., Fix M, Brüggemann O., Electrophoresis, 1997, 10, 1899-905.
[2] Rovio S., Yli-Kauhaluoma J., Siren H., Electrophoresis, 2007, 28, 3129.